The present invention relates to novel fluorescein derivatives, to liposome preparations involving the same and to immunoassay systems based on the use of such liposomes.
Liposomes are micron-sized spherical shells of amphipathic molecules which isolate an interior aqueous space from the bulk exterior aqueous environment. They can be made to contain hydrophobic molecules within their membrane, or hydrophilic markers within their internal aqueous space, or both. This versatility makes liposomes of interest both as potential vehicles for the delivery of drugs in vivo and as the basis for homogeneous immunoassay systems in vitro.
Several immunoassay systems utilizing liposomes have been described. For example, O'Connell, et al., Clin. Chem., 31:1424-1426 (1985) describe a simple competitive binding immunoassay for detecting digoxin using liposomes encapsulating Sulforhodamine B as tracers. Another immunoassay system is the sensitive, homogeneous Liposome Immuno-Lytic Assay (LILA) which involves the antibody-triggered complement-mediated lysis of liposomes. In an exemplary assay format, a liposome encapsulating a marker is made immunoreactive by coupling, e.g., an antigen to the liposome surface, and is incubated with a fluid sample to be analyzed for the presence of antibodies immunoreactive with the antigen. The subsequent binding of a specific antibody to that antigen forms a liposome immune complex. Upon the addition of serum to this liposome complex, complement activation is initiated leading to lysis of the liposome and release of the internal marker substance.
Detection of this lytic event can be achieved in a variety of ways depending upon the nature of the marker initially encapsulated within the liposome. For example, Kataoka, et al., Eur. J. Biochem., 24:123 (1971) describe sensitized liposomes which release trapped glucose marker when incubated with an appropriate anti-serum and complement source.
Numerous fluorescent markers have been successfully associated with or encapsulated within liposomes including both lipid-soluble compounds such as 1,6-diphenyl hexatriene, diacyl oxacarbocyanine, diacyl indocarbocyanine and 4-nitrobenzene 2-oxadiazole; and water-soluble compounds such as carboxyfluorescein, lucifer yellow, aminonapthalene trisulfonate and anilino-napthalene sulfonate, Barbet, J., Fluorescent Tracers for Liposomes. Inserm, 107: 27-36 (1982).
Various methods for the fluorescent detection of liposome lysis have been described including the encapsulation of a fluorophore at self-quenching concentrations followed by lysis and re-establishment of fluorescence, Weinstein, et al., Science, 195:489-491 (1977); dilution of a fluorophore and quencher Vanderwerf, et al., Biochem. Biophys. Acta., 596:302-314 (1980), fluorescent complex formation, Wilschut, et al., Biochemistry, 19:6011-6021 (1980); quenching by complex formation, Kendall, et al., J. Biol. Chem., 257:13892-5 (1982); and resonance energy transfer using two fluorophores, Struck, et al., Biochemistry, 20:4093-4099 (1981).
Where a fluorescent compound is encapsulated at self-quenching concentrations within the interior aqueous space of the liposome, upon liposome lysis an extreme dilution of the fluorophore occurs. A self-quenching concentration is defined to be that concentration at which the fluorescence of the fluorophore has been reduced relative to the fluorescence maximally attainable under conditions of extreme dilution. Subsequent dilution re-establishes fluorescence and the increase in fluorescence over background levels is, ideally, proportional to the concentration of the analyte present in the assay sample.
As one example, Ishimori, et al., J. Immuno. Methods, 75:351-360 (1984) describe an immunoassay technique using immunolysis of liposomes to measure antibody against protein antigens such as human IgG. The release marker used is carboxyfluorescein and the technique is assertedly effective at detecting 10.sup.-15 mole of anti-human IgG antibody, or, in an inhibition assay, human IgG. Yasuda et al., J. Immun. Methods, 44:153-158 (1981), describe the utilization of complement-mediated immune lysis of fluorescent dye encapsulating liposomes to measure anti-glycolipid antibody. Multilamellar liposomes containing carboxyfluorescein, self-quenched at high concentrations, are prepared and upon addition of anti-glycolipid serum plus active complement, liposome lysis occurs and trapped carboxyfluorescein is released.
The fluorophores most commonly used in liposome studies, fluorescein, carboxyfluorescein, and calcein, all show appreciable leakage over a short time scale of hours to days. Weinstein, et al., Science, 195:489-492 (1977), studying liposome-cell interactions, report that the half-time of fluorescein leakage at 5.degree. C. is about 5 minutes whereas that of 6-carboxyfluorescein, a more polar derivative of fluorescein, is on the order of weeks.
Although carboxyfluorescein is less permeable than fluorescein across liposome membranes, the fluorescent yield of carboxyfluorescein is highly dependent upon pH and only the tri-anionic form has maximal fluorescence. The ionic strength, calcium concentration, and temperature of the solution have an influence on fluorescence. Moreover, Lelkes, P. et al., Biochim. et Biophys. Acta., 716:410-419 (1982), investigating the stability of small unilamellar liposomes in human blood, report the need to correct for detergent and centrifugation effects as well as for passive liposome-blood cell association. The use of certain detergents resulted in strong fluorescence quenching and centrifugation of liposomes resulted in a 7-8.8% release of carboxyfluorescein from the liposomes. The addition of approximately 50 mol% cholesterol was required to significantly increase liposome stability. While carboxyfluorescein is highly fluorescent and has a lower leakage rate, fluorescent compounds that are more polar or more strongly ionic are often preferred because of their insensitivity to pH changes. Insensitivity to changes in pH is particularly advantageous because in those assay systems requiring complement lysis of liposomes, the pH may be readily adjusted for optimum complement activity and optimum assay sensitivity without affecting fluorophore leakage or signal.
The fluorescence of calcein, a more electronegatively charged derivative of fluorescein, is largely pH-independent over the pH range of 6.0-8.5. Leakage of calcein from a variety of phospholipid vesicles, as a function of temperature, and in the presence and absence of human serum is reported in Allen, T.M. et al., Biochim. et Biophys. Acta., 597:418-426 (1980). The presence of serum signifanctly increased liposome leakage and the incorporation of increasing molar ratios of cholesterol into liposomes was required to reduce leakage of calcein from liposomes incubated with buffer and with serum. Leakage was significantly higher from liposomes with an osmotic gradient across the membrane (higher inside) than from equiosmolar liposomes.
Calcein can be an acceptable fluorophore in terms of leakage for those applications where the experiments are run the same day. However, it is frequently unacceptable for other applications due to its finite leakage and its susceptibility to quenching by metal ions, i.e., the chelating groups on the xanthene ring bind a large number of different metal ions, Kendall, et al., Analytical Biochemistry, 134:26-33 (1983). Calcein fluorescence is quenched by the binding of Fe.sup.2+, Fe.sup.3+, Ni.sup.3 +, Mn.sup.2 +, Co.sup.2+ and Tb.sup.3 +. These metals can be common contaminants in water and in laboratory glassware. Since lysis in a liposome immunoassay is detected by the release of liposome contents and resultant increase in fluorescence, any quenching of fluorescence leads to an inaccurate measure of lysis and consequently an inaccurate assay result.
There continues to exist a need in the art for fluorescent compounds suitable for use in the preparation of fluorophore-encapsulated liposomes for lytic assays, including immunolytic assays, involving release of fluorophores maintained at self-quenching concentrations within the liposomes. Compounds of this type would be chemically stable and readily synthesized and purified. Ideally they would have a fluorescence spectrum similar to that of fluorescein (allowing use of existing fluorescence detection apparatus based on fluorescein spectral characteristics) and would provide quantum yields at around neutral pH which are no less than about 80% based on maximal fluorescein fluorescence. The compounds should have solubility characteristics allowing for encapsulation at self-quenching concentrations, yet have few ionizable groups in order to minimize osmotic effects. When projected for use in analytical systems wherein metal ions comprise part of the sample milieu, the fluorescence characteristics of the compounds should be minimally sensitive to such ions. The compounds should be susceptible to minimal leakage across liposome membrane layer(s).